Many proteins that have been identified and isolated from human and animal sources have been found to show promising medicinal or therapeutic potential. Great strides have been made in the methods for identifying and characterizing such proteins, in addition to methods for producing such proteins in relatively pure forms and relatively large quantities. As the development process advances in relation to the utilization of such potentially valuable materials many obstacles have arisen in formulating these compounds for use in clinical models.
For example, many such proteins have been found to have an extremely short half life in the blood serum. For the most part, proteins are cleared from the serum through the kidneys. The systematic introduction of relatively large quantities of proteins, particularly those foreign to the human system, can give rise to immunogenic reactions that, among other problems, may lead to rapid removal of the protein from the body through formation of immune complexes. For other proteins, solubility and aggregation problems have also hindered the optimal formulation of the protein.
One of the most promising techniques for addressing these problems has been covalently bonding one or more inert polymer chains to the polypeptide of interest. The most commonly used polymer is polyethylene glycol (PEG), or monomethoxyl polyethylene glycol (mPEG). See, for example, Davis et al., Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, pp. 441-451 (1980). PEG is ideal for these purposes due to its proven non-toxic properties. Other researchers have utilized polyoxyethylated glycerol (POG) for similar purposes. Knauf et al., J. of Biolog. Chem. vol. 263, pg. 15064 (1988).
Numerous results have been described whereby the covalent modification of proteins with polyethylene glycols (“pegylation”) have resulted in the addition of desirable characteristics to the protein. For example, the pegylation of IL-2 has been shown to decrease the clearance of IL-2 while not significantly affecting the activity of the cytokine. The decreased clearance leads to an increased efficiency over the non-pegylated material. Katre et al., Proc. Natl. Acad. Sci. U.S.A. vol. 84, pg. 1487 (1987).
Increasing the half-life of Superoxide Dismutase (SOD) in blood serum has been a critical barrier for the use of SOD for the treatment of various symptoms. A number of studies have shown that the pegylation of SOD will give rise to a decreased clearance rate. See, for example, Conforti et al., Pharm. Research Commun. vol. 19, pg. 287 (1987).
Aggregation of Immunoglobulin G (IgG) has been postulated as a factor that leads to serious side effects to patients that are intravenously administered IgG. It has been shown that the pegylation of IgG reduces the aggregation of the proteins to prevent this problem. Suzuki et al., Biochem, Biopys. Acta vol. 788, pg. 248 (1984).
The ability of pegylation techniques to affect protein immunogenicity has also been shown. Abuchouski and coworkers have studied the immunogenicity and circulating life of pegylated Bovine Liver Catalase. Abuchowski et al., J. Biol. Chem. vol. 252, pg. 3582 (1977).
The addition of PEG groups to these various proteins decreases clearance due to the increase in molecular size of the pegylated protein. Up to a certain size, the rate of glomerular filtration of proteins is inversely proportional to the size of the protein. The ability of pegylation to decrease clearance, therefore, is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protien. This has been borne out by clearance studies that varied both the size of the PEG side chains and the number of PEG units bonded to IL-2. Katre, supra.
The various studies of pegylated proteins in relation to clearance, immunogenicity, aggregation and physical properties all suggest that the PEG forms a flexible, hydrophilic shell around the protein. The PEG chains become highly hydrated and give the pegylated proteins a higher apparent molecular weight than would be predicted, and act to shield charges on the protein.
Because of the many promising results that have been seen in this field, a catalogue of procedures for the attachment of PEG units to polypeptides has been developed. The key element in these procedures is the “activation” of the terminal—OH group of the polyethylene glycol. Such activation is necessary in order to create a bond between the PEG group and the polypeptide. The vast majority of coupling procedures activate the PEG moiety in order to react with free primary amino groups of the polypeptides. Most of these free amines are found in the lysine amino acid residues.
In general practice, multiple PEG moieties are attached to the proteins. For example, in U.S. Pat. No. 4,179,337 of Davis et al., it was found that to suppress immunogenicity it is desireable to use between 15 and 50 moles of polymer per mole of polypeptide.
Because multiple PEG chains are generally bonded to each polypeptide, and because there are typically a large number of lysine residues in each protein, there has been little effort to pegylate proteins to yield homogenous reaction products. See, Goodson et al. Biotechnology, vol. 8, pg. 343 (1990); U.S. Pat. No. 4,904,584 of Shaw. This lack of reaction specifity gives rise to a number of complications. Among these, are that pegylation often results in a significant loss of activity of the protein. Presumably, attachment to a critical lysine residue could alter the active site of the protein rendering it inactive.
It has been shown in at least one system, that pegylation can lead to sterically hindered active sites. In other words, relatively small substrates may approach the protein, while the activity of proteins that react with larger substrates can be dramatically effected by random pegylation. Davis et al. supra. The site selective pegylation of such proteins could lead to modified materials that gain the desireable attributes of pegylation without the loss of activity. In addition, if the pegylated protein is intended for therapeutic use, the multiple species mixture that results from the use of non-specific pegylation leads to difficulties in the preparation of a product with reproducible and characterizable properties. This makes it extremely difficult to evaluate therapeutics and to establish efficacy and dosing information.
In certain cases, it has been found that the administration of multimeric complexes that contain more than one biologically active polypeptide or drug can lead to synergistic benefits. For example, a complex containing two identical binding polypeptides may have substantially increased affinity to the ligand or active site that it binds relative to the monomeric polypeptide. For this reason, multimeric complexes of proteins can be desirable in order to increase affinity of the protein to its ligand in addition to increasing the molecular weight of the complex.
Proteins frequently achieve their biological effects through interaction with other proteins. Where a simple complex of two proteins is sufficient to achieve the biological effect it has proved possible to mimic the physiological effects of endogenous proteins by administering exogenous proteins. However, where the biological effect requires the assembly of a complex containing more than two proteins it is more difficult to mimic the function of the endogenous proteins with recombinantly produced exogenous equivalents because the higher order complexes are frequently unstable. In such cases it may be advantageous to use crosslinked species containing two of the components of the complex to simulate the biologically-active complex.
Subsequent to the invention described herein, at least three research groups have described the production of crosslinked proteins, where the extracellular portions of one of the TNF receptors is attached to the heavy chain of human or mouse IgG, which are then crosslinked through disulfide bonds. Peppel et al., J. Exp. Med. vol. 174, pg. 1483 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci. USA vol. 88, pg. 10535 (1991); and Loetscher et al., J. Biol, Chem. vol. 266, pg. 18324 (1991). In each case, the proteins were expressed in animal cell expression systems, and were found to be substantially more effective at inhibiting TNF than the monomeric soluble receptor alone. Similar procedures have also been used for producing similar crosslinked proteins of the CD4 protein, (Byrn at al., Nature (London) vol. 344, pg. 667 (1990)) the CR1 protein, (Kalli et al., J. Exp. Med. vol. 174, pg. 1451 (1991); Hebell et al., WO 91/16437 (1991)) and the CR2 protein. (Hebell et al., Science, vol. 254, pg. 102 (1991)).
These crosslinked proteins—constructed of two polypeptide units and a portion of the IgG antibody—have been shown to have promise as therapeutic agents. The crosslinked proteins have an increased molecular weight, which acts to decrease the rate of clearance of the complex from the body, in addition to the apparent enhancement of the affinity of the proteins to their ligand. However, the proteins crosslinked in this manner have so far only been prepared by expression in animal cell expression systems by the expression of fused genes. This has been required in order to have the IgG portion of the protein properly folded after expression. In addition, the fixed heavy chain portion of the IgG antibody that serves as the spacer or linker between the polypeptide units does not allow for the ability to vary the length, size or geometry of the spacer. Given the apparent synergistic effect achieved by the dimeric proteins, it is likely that by varying the spatial orientation of the polypeptides the synergistic benefit may be optimized. And finally, the crosslinked proteins may be antigenic and/or have decreased solubility. The heavy chain of antibodies is not biologically inert.
Other dimeric or “bivalent” complexes have been described. One such group of dimeric compounds has been labeled hirulogs. These compounds are comprised of very short polypeptide units that are linked by a short poly-glycine spacer or linker. One of the polypeptide units is a thrombin inhibitor—a 5 amino acid sequence taken from the 65 amino acid protein Hirudin—and the other is an anion-binding exocite (ABE) recognition inhibitor. See, Maragonore et al., Biochemistry, vol. 29, pg. 7085 (1990); Bourdon et al., FEBS vol. 294, pg. 163 (1991).
C-reactive protein (CRP) is an acute phase serum protein composed of five identical 23 kDa subunits. CRP can induce reactions of precipitation and agglutination and can also react with Clg to activate the classical complement pathway. Cross linked oligomers of CRP have been formed using bis (sulphosuccinimidyl) suberate or 3,3′-dithio (sulphosuccinimidylpropionate) as cross-linking agents. Jiang et al., Immunology vol. 74, pg. 725 (1991).
The formation of dimeric or bivalent ligands for targeting opoid receptors has also been investigated. Non-peptidic β-naltrexamine or oxymorphamine pharmacophores have been connected by short ethylene oxide or glycine spacers. Erez et al., J. Med. Chem. vol. 25, pg. 847 (1982); Portoghese et al., J. Med. Chem. vol. 29, pg. 1855 (1986). Tetrapeptide enkephalins linked by short methylene bridges have also been designed to target opoid receptors, and have been shown to have a greater selectivity and affinity for the delta receptor than the original delta ligand. Shimohigashi et al., Nature vol. 197, pg. 333 (1982).
The cell surface glycoprotein CD4 has also been produced in multimeric forms through a sugar-based cross-linking strategy. The cross-linking agent utilized was bismaleimidohexane (BMH). Chen et al., J. Biol. Chem. vol 266, pg. 18237 (1991).
Lymphocyte function-associated antigen-3 (LFA-3) is a widely distributed cell surface glycoprotein that is a ligand for the T lymphocyte CD2. LFA-3 with its associated lipids forms protein micelles of eight monomers which increased their ability to interact with cells with CD2 on their surface. Dustin et al., J. Exp. Med., vol. 169, pg. 503 (1989).
In a somewhat related technology, one group has studied the inhibitory effect of a synthetic polypeptide that is comprised of a repeating pentapeptidyl unit. The polymer was synthesized by the polymerization procedure with diphenyl phosphoryl azide to a size of about 10,000 daltons. The polymerized pentapeptide is one of the essential structures in several biological responses. Morata et al., Inst. J. Biol. Macromol. vol. 11, pg. 97 (1989).
A further obstacle in developing effective exogenous proteins to augment or compete with endogenous substances is that exogenous proteins must be administered systematically rather than being localized in the appropriate place. This can lead to lower efficacy and to increased side effects. Several groups have reported targeting bioactive proteins to the appropriate sites by linking them to other proteins that naturally home on those sites. Often such linkages are made through gene fusions between the active and the targeting proteins.
Polyethylene glycol spacer or linker units have been used to create antibody targeted superantigens after the date of the instant invention. A monoclonal antibody reactive to colon carcinoma cells was attached to the bacterial superantigen staphylococcal enterotoxin. Rather than being designed to exploit the benefits associated with the other bivalent complexes (e.g., higher molecular weight; synergistic effects of bivalency) these complexes are designed to target superantigens to specific locations. The pegylation process described to form these targeted superantigens creates a complex containing a large mixture of materials. The coupling of the antibody and the superantigen was accomplished by the use of N-succin-imidyl 3-(2-pyridyldithio) proprionate and a 24-atom-long PEG-based hydrophilic space. According to this procedure 7 to 18 spacers were attached to each antibody unit and one or two lysines on each of the super antigens were reacted. Dohlsten et al., Proc. Natl. Acad. Sci. USA vol. 88, pg. 9287 (October, 1991). Using this procedure it would be impossible to isolate a single species in order to optimize the product or process.
Two groups of proteinaceous materials having significant applications for the treatment of a wide variety of medical indications are Tumor Necrosis Factor (TNF) inhibitors and Interleukin-1 receptor antagonists (Il-1ra). These materials have been shown to have beneficial effects in the treatment of TNF and IL-1 mediated diseases respectively. Among the indications that have been identified as being either TNF mediated or IL-1 mediated, are Adult Respiratory Distress Syndrome, Pulmonary Fibrosis, Rheumatoid Arthritis, Inflammatory Bowel Disease and Septic Shock.
Copending U.S. patent application Ser. No. 07/555,274, filed Jul. 19, 1990, specifically incorporated herein by reference, describes a class of naturally occurring proteinaceous TNF inhibitors and a method for manufacturing a substantial quantity of the same with a high degree of purity. In particular, the aforementioned application describes in detail two subsets of TNF inhibitors referred to as 30 kDa TNF inhibitor and 40 kDa TNF inhibitor. In addition to the full-length 40 kDa TNF inhibitor protein, two truncated, yet biologically-active, forms of the 40 kDa TNF inhibitor have also been produced. These proteins, in which 51 and 53 carboxyl termini amino acids have been removed from the full-length protein, are referred to respectively as 40 kDa TNF inhibitor Δ51 and 40 kDa TNF inhibitor Δ53.
Copending U.S. patent application Ser. No. 07/506,522, filed Apr. 6, 1990, specifically incorporated herein by reference, describes a preferred class of naturally occurring, proteinaceous Il-1 inhibitors and a method for manufacturing a substantial quantity of the same with a high degree of purity. In particular, the application describes in detail three such interleukin-1 inhibitors which are interleukin-1 receptor antagonists (IL-1ra's), namely, IL-1raα, IL-1raβ, and Il-1rax.
Two additional classes of materials that are potentially useful for the treatment of a variety of medical indications are interleukin-2 inhibitors and complement inhibitors. Potential inhibitors of interleukin-2 include interleukin-2 receptors, the extracellular portion of interleukin-2 receptors, interleukin-2 receptor antagonists, antibodies that recognize interleukin-2, and fragments of any of such species that contain the IL-2 binding function. Potential inhibitors of the complement system include the receptor CR1, the extracellular portion of CR1, and the fragment of CR1 that contains the complement binding function.
Interleukin-2 receptor has been described and methods for its isolation have been disclosed in U.S. Pat. No. 4,578,335 of Urdal et al. and U.S. Pat. No. 4,816,565 of Honjo et al. The gene encoding Interleukin-2 receptor and methods for its recombinant production have also been disclosed. European Patent Application No. 89104023.0 of Taniguchi et al.; European Patent Application No. 90104246.6 of Taniguchi et al. See also, Honjo et al., Nature vol. 311, pg. 631 (1984); Taniguchi et al., Science vol. 244, pg. 551 (1989).
It could be assumed that to some extent the soluble extracellular domain of either interleukin-2 receptor will act as an inhibitor to the action of the cytokine interleukin-2. Interleukin-2 is one of the best characterized cytokines, known to play a pivotal role in the antigen-specific clonal proliferation of T lymphocytes. Interleukin-2 has also been shown to act on a variety of other cells in the immune system.
There are three discrete forms of the interleukin-2 receptor, comprised of two distinct receptor molecules designated either as IL-2rα and IL2rβ.
The highest affinity IL-2 receptor is composed of two distinct IL-2 receptors. Both of these receptors have been cloned and characterized. The low affinity IL-2 receptor (IL-2rα) was cloned in 1984 and has been well characterized. Nikaido et al., Nature vol. 311, pg. 631 (1984). The extracellular domain of the molecule has a molecular weight of 24,825 and has two N-linked glycosylation sites. The molecule contains 11 cysteines, 10 of which are involved in intramolecular disulfide bonds. The putative IL-2 binding domains on the molecule have been mapped both by mutagenesis and epitope mapping.
The intermediate affinity IL-2 receptor (Il-2rβ) was cloned in 1989 and has not been as completely characterized as IL-2rα. Hatakayama et al., Science vol. 244, pg. 551 (1989). The extracellular domain of IL-2rβ has a molecular weight of 24,693. The molecule contains 8 cysteines and 4 N-linked glycosylation sites. The disulfide bonding in the molecule is unknown. IL-2rβ has a cytoplasmic domain of 286 amino acids.
The disassociation constants (Kd's) for the IL-2 receptors have been determined. They are 10−8M for IL-2rα, 10−9M for IL-2rβ and 10−11M for the high affinity receptor which consists of a complex of IL-2rα, IL-2rβ and IL-2. Current models indicate that the formation of the high affinity complex is formed first by IL-2 binding to IL-2rα and then to IL-2rβ. Ogura et al., Mol. Biol. Med, vol. 5, pg. 123 (1988).
An inhibitor of IL-2 may be valuable in the prevention of transplant rejection as well as autoimmune disorders. Currently, a monoclonal antibody against IL-2rα that prevents IL-2 binding is being tested in human renal transplantation. Hiesse et al., La Presse Mediocle vol. 20, pg. 2036 (1991). In a study of 15 patients, the antibody, in combination with immunosuppressants, has been shown to be as effective in preventing allograft rejection as a control group getting higher doses of immunosuppressants. High levels of circulating soluble IL-2rα have been detected in a number of diseases, some infections, as well as transplantation and rejection. This suggests involvement of IL-2 in these diseases.
CR1 is a protein also referred to as the C3b/C4b receptor. CR1 is present on erythrocytes and a variety of other cell types, and specifically binds C3b, C4b, and iC3b. CR1 can also inhibit the classical and alternate pathway C3/ C5 convertases and act as a cofactor for the cleavage of C3b and C4b by factor 1. Fearon et al., Proc. Natl. Acad. Sci. USA vol. 75, pg. 5867 (1979). CR1 is a glycoprotein composed of a single polypeptide chain, and there are four allotypic forms. It is known that CR1 contains repetitive coding sequences, and this fact is used to explain the existence of multiple allotypes. Krickstein et al. Complement vol. 2, pg. 44 (Abst.) (1995).
The diminished expression of CR1 on erythocytes has been associated with systemic lupus erythematosus and CR1 number has also been found to correlate inversely with serum level of immune complexes. The CR1 protein, the CR1 gene and methods for the production of CR1 are described in WO 91/05047 and WO 89/09220 of Fearon et al. As described above, dimeric species containing CR1 and portions of an antibody have also been disclosed. WO 91/16437 of Hebell et al.